Cellulose is produced by plants and by various microorganisms. Examples of cellulose producing prokaryotic organisms include Acetobacter, Rhizobium, Agrobacterium and Aztobacter. Harris et al., (1973) Ann Rev Microbiol 27:27-50. Acetobacter is one of the best characterized cellulose producing organisms.
Acetobacter is characteristically a Gram-negative, rod-shaped bacterium 0.6-0.8 um by 1.0-4 um. It is strictly aerobic, metabolism is respiratory, never fermentative. It is further distinguished by its ability to simultaneously produce multiple poly-.beta.(1-4) glucan chains, which are chemically identical to cellulose. Multiple cellulose chains or microfibrils are synthesized at sites on the bacterial cell wall. These microfibrils are extruded by the bacterium into the culture medium.
The cellulose microfibrils produced by Acetobacter have cross-sectional dimensions of about 1.6 nm.times.5.8 nm. In static or standing cultures, the microfibrils at the bacterial surface combine to form a fibril having cross-sectional dimensions of about 3.2 nm.times.133 nm. This small cross-sectional width is the chief difference between cellulose produced by Acetobacter and cellulose found in wood pulp. The small cross-sectional size of these Acetobacter-produced fibrils, together with the concomitantly greater surface area than conventional wood-pulp cellulose and the inherent hydrophilicity of cellulose, leads to a cellulose product having unusually great capacity for absorbing aqueous solutions. This capacity for high absorbency has led to the development of several products from Acetobacter cellulose, such as wound dressings and paper products.
In 1947, Hestrin et al, (1947) Nature 159:64-65 found that in the presence of glucose and oxygen, non proliferating cells of Acetobacter synthesize cellulose. Since that time, Acetobacter has been cultured under a variety of conditions which support cellulose production. For example, when grown with reciprocal shaking at about 90-100 cycles per minute, cells have been incorporated into a large gel mass. When grown under conditions in which the culture medium is agitated with swirling motion for four hours, stellate gel bodies form which are comprised of cellulose and cells. When grown as standing cultures, a pellicle forms at the air/medium interface. The pellicle forms as a pad generally having the same surface shape and area as the liquid surface of the vessel containing the culture. Hestrin and Schramm, (1954) Biochem J 58:345-352.
In static cultures, production of cellulose by Acetobacter occurs at the air-liquid medium interface. Acetobacter is an obligate aerobe, therefore each bacterium continuously produces one fibril at the air-liquid interface. As new cellulose is formed at the surface, existing cellulose is forced downward into the growth medium. As a result, cellulose pellicles produced in static culture conditions consist of layers of cellulose fibers which support the growing Acetobacter cells at the air medium interface. Significantly, the volume of cellulose so produced is restricted by the interface between air and culture medium.
Acetobacter also produce cellulose when grown in agitated cultures. However, for several reasons, high cellulose productivity from agitated cultures has previously been difficult to attain. The tendency of known Acetobacter strains to become non-producers when cultured under agitated conditions at increased dissolved oxygen concentration has in the past severely limited the amount of cellulose that can be made economically. U.S. Pat. No. 4,863,565 discloses a number of strains of Acetobacter that are stable in long term cultures under both agitated and static conditions. That patent describes selected Acetobacter strains characterized by a sharply reduced ability to form gluconic and keto-gluconic acids when grown on a glucose containing medium. The strain used in the present invention, strain 1306-21, is one of the stable strains disclosed in that patent.
A second problem that has limited cellulose production in agitated cultures stems from the high agitation rates required to oxygenate the fermentation medium as the cellulose and cell concentrations of the culture increase. Oxygen demand of a culture increases as the cell density of the culture increases. Further, at high cellulose concentrations the fermentation broth becomes more viscous, thus the oxygen transfer rate from the air phase to the liquid phase decreases. Therefore, increasing agitation rates are required to sustain sufficient levels of dissolved oxygen in the culture to maintain cell growth and cellulose production. However, the increased agitation negatively affects the amount of cellulose produced due to a substantial decrease in cellulose yield and volumetric productivity.
Cellulose production by Acetobacter has been shown to decrease as the agitation rate of the culture is significantly increased (see, for example, the data provided in Example 1, infra). Increasing the agitation rate may adversely affect the cellulose to cell ratio and/or the cellulose yield of the culture. These effects may be attributable to increasing shear stress on the cells and cellulose as the agitation rate of the culture is increased. At high agitation rates, the quality of the cellulose produced is also affected. See, for example, U.S. Pat. No. 4,863,565 which discloses that in tests in a fermentor (14 liters) using an impeller to agitate the broth, it was found that the characteristics of the broth (viscosity) and the resulting cellulose (particle size and morphology, settling rate, hand sheet formation) were affected by high impeller speeds (above about 600 rpm in the runs carried out). These effects were more pronounced the longer the cultures were agitated at such speeds.
The present invention discloses the use of polyacrylamides and closely related anionic copolymers. The closely related anionic copolymers are formed from combined polymerization of acrylamide and acrylic acid (or its salts) or partial hydrolysis of amide functional groups of a polyacrylamide.
Polyacrylamides are used for many different purposes. They are commonly used as flocculating agents to clarify liquids. Some of the uses of polyacrylamides include sludge thickening, polymer recovery, crystal control, and use as aids in dewatering. The compounds can also be used as aids in vacuum filtration and centrifugation to increase production rate and cake solids capture.